Gel electrophoresis is an important technique widely used for separating biological molecules by molecular weight. At present, restriction fragment analysis of nucleic acid using gel electrophoresis is predominantly used as (1) a qualitative technique, e.g., for assessing the success of a particular cloning step in terms of the presence or absence of specific fragments, base pair sequencing, determining the possible presence of impurities, etc., or (2) as a quantitative means of measuring the molecular weight (molecular length) of the separated fragments in terms of their dispersed position. Much less common has been the additional measurement of the mass of separated nucleic acid which can be determined from the intensity of the fluorescence of the stained sample.
The principal method now employed to detect the distribution of DNA or RNA strands separated by gel electrophoresis involves use of a fluorescent dye, e.g., ethidium bromide. Under the proper conditions the sample mass is directly proportional to the fluorescence signal (1, 2). Present methods of measuring the mass distribution in terms of fluorescence (visual estimation, photography/scanning densitometry, electronic imaging) are labor intensive, slow or have one or more of the following drawbacks: limited accuracy, insufficient sensitivity, and restricted dynamic range. These drawbacks have restricted the wider use of mass distribution measurements.
After separation the most common procedure is to expose the stained gel to mid-ultraviolet illumination centered at 310 nm provided by a transilluminator containing one or more low wattage, UV emitting lamps. The resulting sample fluorescence with peak emission in the orange at 590 nm is observed visually or photographed.
The simplest means for estimating the mass of the separated fragments is to visually compare the brightness of the fluorescence of the separated sample bands to that from a reference standard of known sample mass added to an adjacent lane of the gel which separates into a simple pattern of fragments whose individual mass can be closely estimated. The method is inaccurate because the observer cannot easily correct for the differing fluorescence intensities from two samples of equal mass but spread out over different areas.
Densitometer tracings of the photographic negative of the fluorescent band pattern are frequently employed to accurately determine the distances of migration of polydisperse samples. This procedure has important limitations when used to measure the fluorescence intensity to determine the mass of dispersed DNA in each band. The darkening of the photographic negative is linearly proportional to the logarithm of the exposure over a limited exposure range between one and two orders of magnitude. Within this range the fluorescence intensity, I, may be expressed as: EQU I.alpha.10.sup.D /.gamma.,
where D is the optical density (in the range above threshold for linearity) measured by the densitometer, and .gamma. is the contrast index or slope of the linear portion of optical density plotted against log exposure. Since the fluorescence intensity is indirectly derived from a densitometer measurement, it is subject to inaccuracies and noise involved in the measurement of the optical density.
The contrast index of the film can be markedly affected by the development time, and to a lesser extent by the development temperature, storage conditions, and the exposure time (reciprocity failure). The stringent controls which are required to prevent unacceptable variations are inconvenient to maintain in routine analysis. Instead, suitable DNA standards which span the mass range of interest are introduced on every gel to provide a calibration curve. The camera exposure time must be chosen carefully so that the range of exposures closely matches the linear portion of the film characteristic curve to prevent unacceptable flattening of the calibration curve at its ends. This procedure is labor intensive and limits the number of samples which can be prepared and analyzed.
The densitometer measurement time can become unacceptably long. Often the DNA is not distributed uniformly across the lane and an accurate measurement of fluorescence requires sampling at more points than are normally required to measure the lane position. The densitometer sampling spot size must be small compared to spatial variation in density or error will result, further increasing the measurement time. Additional points must also be sampled adjacent to each lane to permit accurately correcting for the baseline fluorescence contributed by the residual free dye which remains after destaining.
The accuracy of the method is also affected by the uniformity of the UV illumination, since the fluorescence is directly proportional to the intensity of the exciting light. Variation across the field-of-view can be 10% or greater using commercial transilluminators. There is also considerable drift in intensity with warmup time. The sensitivity is limited by a high level of background light from several sources unrelated to the stained gel (3).
Electronic imagining of the gel has been employed which measures the fluorescence intensity directly and greatly reduces the measurement time (4). The method has the drawbacks of limited dynamic range, and difficulties in correcting accurately for variation in the illumination over the field-of-view and for pixel-to-pixel sensitivity variation. As with photography, highly accurate measurements of mass are hindered by geometric field distortion, vignetting, and loss of resolution unless the field angle is severely limited, particularly for thick sample gels. Reduced signal-to-noise ratio at low light levels has led to little improvement in sensitivity over the photographic technique.
CW lasers have been employed as excitation sources in tube gels (5) and most recently with slab gels in various automated DNA sequencers which detect separated DNA fragments labelled with fluorophores specific to the base pair terminating the fragment (6-11). Large improvement in sensitivities has been reported for polyacrylamide gels and dyes which differ from ethidium bromide. However, there are no reports for the use of such instruments for measurement of nucleic acid mass using ethidium bromide in either polyacrylamide or agarose gels and the descriptions of their design do not include the unique performance requirements for that application.